Myosin is found in all eukaryotic cells and appears to be involved in diverse cellular motile processes such as cytokinesis. This laboratory has isolated two different cDNAs for nonmuscle myosin heavy chains (MHCs), MHC-A and MHC-B, which are encoded by two different genes. Previously, we have demonstrated tissue and cell type-dependent expression for the two MHCs. In this study, we have been identifying the cis-regulatory elements that are responsible for the cell type-dependent transcription in the nonmuscle MHC-A gene that results in MHC-A mRNA being abundantly expressed in connective tissue cells (fibroblasts and glial cells) and proliferating myoblasts, but less abundantly expressed in neuronal cells and differentiated muscle cells. We examined the region extending approximately 20 kb upstream and approximately 40 kb downstream from the transcriptional start sites, using transient transfection analysis of the luciferase reporter constructs. A 100 bp region, located 23 kb downstream from the transcriptional start sites, in intron 1 has been found to activate transcription in NIH 3T3 fibroblasts and proliferating C2C12 myoblasts, but not in differentiated C2C12 myotubes. Gel shift assay and methylation interference analysis using NIH 3T3 nuclear extracts has revealed the existence of three clustered binding elements for transcriptional factors. The most 5' element C, a G-rich sequence, is recognized specifically and exclusively by Sp1 and Sp3, and is immediately followed by a novel element A. The third element F, which is located 50 bp 3' to the above elements, contains an E-box sequence (CANNTG) and can be recognized partially by USF (Upstream Stimulatory Factor) 1 and/or 2. Transfection studies with luciferase reporter constructs which include mutations in all three elements in various combinations demonstrate that elements A and C cooperatively activate transcription. Element F shows an additive effect. Mutations in all three elements abolishes transcriptional enhancement activity almost completely.